Phosphoinositides (PIPs) are a group of inter-convertible lipid messengers harboring single, double or triple phosphoryl moieties on positions 3, 4 and/or 5 of their inositol ring. Although they account for a minor fraction of total cellular lipids, PIPs appear to be major second messengers in signaling networks by tyrosine kinase receptors of hormones and growth factors, by several G-coupled receptors, as well as by environmental cellular stresses. Bursts of increase in PI-3,4,5-P3 (PIP3), largely produced by 3′ phosphorylation of PI-4,5-P2 (phosphatidylinositol 4,5-bisphosphate) in a process catalyzed by phosphatidylinositol 3-kinase (PI 3-kinase), is a major signaling step mediating cellular functions as diverse as metabolism, cellular motility, gene regulation and cell survival (anti-apoptosis).
One example for PIPs involvement in mediating cellular function is its role in diabetes. Diabetes type II is characterized by insulin resistance which is a state of impaired metabolic response to insulin. Current pharmacological therapeutics is mostly based on activation or inhibition of enzymes or receptors, a strategy somewhat limited when the disease process involves impaired insulin signaling capacity. Phosphatidylinsoitol-3,4,5-trisphosphate (PIP3), production induced by insulin, has now been shown to constitute a major lipid messenger in insulin-mediated metabolic actions in muscle and fat. Intracellular delivery of PIP3 into cells would therefore be ideal for bypassing the signaling defect.
Altered skin wound healing is a common cause of morbidity and mortality among diabetic patients, caused in response to a rise in blood sugar concentrations. Abnormally high levels of glucose were found to affect the proliferation and differentiation processes of keratinocytes, thereby altering their function. For healing a wound, the cells surrounding the wound must survive, proliferate, migrate and grow directionally to close the wound. The signaling pathway induced by injury involves PIP3 and has been shown to be essential for directional migration of corneal and skin epithelial cells, subsequently promoting wound healing.
Autophagy is another example for PIPs involvement in mediating cellular function. Autophagy, an intra-cellular process in eukaryotic cells, allows for the digestion and recycling of cytoplasmic contents through the formation of double-membrane vesicles (autophagosomes) that undergo degradation through fusion with lysosomes. Basal autophagy plays an important role in cellular homeostasis. Autophagy can also be induced as a cellular reaction to various situations, such as nutrient starvation or pathogen infection. Thus, dysfunction in autophagy has been implicated in the pathogenesis of various diseases, like cancer, infectious diseases and neurodegenerative disorders.
For example, since decrease in autophagy flux is correlated with insulin resistance of hepatocytes in obesity, it may be solved by up-regulating the autophagy flux. It has been suggested that obesity which is correlated with elevated triglycerides, free fatty acids and glucose concentrations in the plasma results in defective autophagy in the liver, which promotes elevated endoplasmic reticulum stress and impaired insulin signaling.
Since autophagy is a dynamic cellular mechanism, in which autophagosomes are being formed and degrade constantly, in order to assess the effect of the complexes on autophagy, the autophagic flux is monitored. The autophagic flux is evaluated by the difference in LC3-II (autophagosomes marker) levels between cells treated with and without bafilomycin A1 (lysosomal fusion/degradation inhibitor). Autophagy is a highly evolutionarily conserved intracellular catabolic mechanism. As such, it is a highly regulated mechanism. One of the regulators of autophagy is calcium which acts as an intracellular second messenger and controls diverse cellular functions. It has been shown that autophagy also regulates Ca2+ mobilization. Therefore, autophagy and calcium mobilization are interrelated and can affect each other.
Autophagy is a multistep mechanism consisting of the initiation of the autophagosome membrane, elongation, maturation and fusion with lysosome for degradation. Stimulation of the vacuolar protein sorting 34 (VPS34) complex generates the local production of a pool of phosphatidylinositol-3-phosphate (PI3P), which promotes autophagosomal membrane nucleation. Since phosphatidylinositol-3-phosphate (PI3P) mediates autophagosome biogenesis through membrane deformation and elongation, it can act as an autophagy activator.
Although PIPs are attractive candidates for therapeutic purposes, such applications are challenged, inter alia, by the need to overcome permeability barriers through the plasma membrane, and by the poor stability of the PIPs. Exogenous PIPs are currently introduced into cells using permeable derivatives, or assisted by polycationic carriers such as polyamines. However, the widely-used carriers, e.g. polyethyleneimine (PEI) suffer from toxicity, poor efficiency, and low biodegradability (Fischer, 1999, Godbey, 2001).